Enzyme inhibiting peptide derivatives

ABSTRACT

Disclosed herein are peptide derivatives which inhibit the activities of human immunodeficiency virus (HIV) protease and renin. The peptide derivatives can be represented by general formula R 1  --R 2  --Y--R 3  --R 4  wherein R 1  is a derived amino acid based on an O 4  -(carboxymethyl)-tyrosyl residue, R 2  and are amino acid or analogous amino acid residues (R 3  may optionally be absent), Y is a non-peptide linking unit, e.g. statyl, and R 4  is [--NR 17  CHR 18  --C(O)] p  --Z wherein R 17  is hydrogen or lower alkyl, R 18  is an amino acid or analogous amino acid side chain, p is zero or one and Z is a terminal group (e.g. hydroxy or amino), or R 4  is --NR 17  CR 18  (R 21 )CH 2  OH wherein R 17  and R 18  are as noted hereinabove and R 21  is hydrogen, lower alkyl or hydroxy(lower)alkyl. The derivatives are useful as agents for combatting HIV infections and for treating renin-associated hypertension and congestive heart failure.

FIELD OF THE INVENTION

This invention relates to compounds having valuable pharmacologicalproperties. More specifically, the invention relates to peptidederivatives (hereinafter called "peptides") exhibiting activity againstparticular retroviruses, to processes for producing the peptides, topharmaceutical preparations thereof, and to a method of using thepeptides to combat infections caused by the retroviruses. The inventionalso includes pharmaceutical formulations and a method of treatment forrenin-associated hypertension and for congestive heart failure whereinthe peptides serve as the active agent.

BACKGROUND OF THE INVENTION

During the last ten years, retroviruses have emerged from relativeobscurity to prominence. These viruses now are known to cause of varietyof diseases in vertebrates, the most insidious to humans beingimmunodeficiencies and cancers.

In 1983, a retrovirus, known as human immunodeficiency virus type 1(HIV-1), was established as a causative agent for acquired immunedeficiency syndrome (AIDS). This virus has become a pestilence ofalarming proportion. More recently, the closely related virus, humanimmunodeficiency virus type 2 (HIV-2) has been identified as a secondcausitive agent of AIDS. (Hereinafter, the term "HIV" is meant toinclude both HIV-1 and HIV-2 and any mutants thereof).

Presently, several compounds are being evaluated in the clinic aspossible therapeutic agents for AIDS. Another compound,3'-azido-3'-deoxythimidine (known also as zidovudine or AZT), has beenshown in the clinic to decrease mortality and the frequency ofopportunistic infections in AIDS patients. This latter compound is beingused to manage certain patients with symptomatic HIV infections.However, in spite of some recent progress, the need for an effectivetherapy for AIDS still exists. For recent reviews, see R. A. Weiss in"Molecular Basis of Virus Disease", Symposium of the Society for GeneralMicrobiology, Vol. 40, Eds. W. C. Russel and J. W. Almond, UniversityPress, Cambridge, UK, 1987, pp 167-192, and R. C. Gallo and L.Montagnier, Scientific American, 259, (4), 40 (1988).

One approach to finding agents having anti-HIV activity is to inhibitthe action of HIV-encoded enzymes. This manner of inhibition interfereswith the replication and propagation of the virus. Such an approach hasbeen applied successfully in a search for inhibitors of the viralencoded enzyme, reverse transcriptase (RT). More explicitly, thepreviously noted zidovudine was found to inhibit RT which is required toeffect viral replication. Subsequently, zidovudine was developed as ananti-HIV agent. Still more recently, this approach has been investigatedusing another HIV-encoded enzyme known as HIV protease as the targetenzyme. In one instance, pepstain A was found to inhibit the intracellarprocessing that provides the requisite HIV protease. See, S. Seelmeieret al., Proc. Natl. Acad. Sci. USA, 85, 6612 (1988). However, thedevelopment of pepstatin A as an anti-HIV agent seems improbable in viewof its multiple activities. In another instance, M. L. Moore et al.,Biochem. Biophys. Res. Comm., 159, 420 (1989), reported oninvestigations showing the inhibition of HIV protease by threeheptapeptide analogs modeled after a conserved cleavage site (gag generegion) of the viral genomic polyprotein. A. D. Richards et al., FEBSLetters, 247, 113(1989), also have reported in vitro inhibition of HIVprotease be acetylpepstatin and a nonapeptide analog.

The present application discloses a group of peptide derivatives whichare potent inhibitors of HIV protease and renin. These attributes,together with the attributes of a relatively selective action and anapparent lack of toxicity, renders the peptides useful as agents forcombating HIV infections and for treating renin-associated hypertensionand congestive heart failure.

The present peptides of this application are distinguished readily frompepstatin A and the previously noted peptide analogs by chemical andbiochemical differences. The present peptides also possess a partialstructural resemblance to peptide derivatives reported to be renininhibitors; for instance, see D. F. Veber et al., European patentapplication 77,028, published Apr. 20, 1983, and A. Wagner et al.,Australian patent application 76241/87, published Feb. 4, 1988. Theremaining structural features and differences in biological profilesdistinguish these latter prior art compounds from the present peptidederivatives, notwithstanding the existence of broad generic disclosures,such as R. Ten Brink, PCT patent application WO87/02986, published May21, 1987, encompassing a myriad of compounds ranging in the millions.Finally, a class of peptide isosteres have been reported recently tohave the unusual combination of renin inhibitory and antiretroviralactivities; the latter compounds have structures which are quitedifferent from the present peptides (see B. Weidmann, UK patentapplication 2203740, published Oct. 26, 1988).

SUMMARY OF THE INVENTION

The peptides of this invention are represented by formula 1

    R.sup.1 --R.sup.2 --Y--R.sup.3 --R.sup.4 1

wherein R¹ is a derived amino acid radical of formula 2 ##STR1## whereinR⁵ is hydrogen, lower alkyl, or R⁷ OC(O)-- or R⁷ C(O)-- wherein R⁷ islower alkyl, lower cycloalkyl, (lower cycloalkyl)methyl, phenyl, phenylmonosubstitued with lower alkyl, lower alkoxy or halo, benzyl or benzylmonosubstituted with lower alkyl, lower alkoxy or halo; R⁶ is hydrogen,lower alkyl, lower cycloalkyl, (lower cycloalkyl)methyl, phenyl, phenylmonosubstituted with lower alkyl, lower alkoxy or halo, benzyl, orbenzyl monosubstituted with lower alkyl, lower alkoxy or halo; L on thearomatic ring of the radical of formula 2 represents hydrogen or asubstituent on the aromatic ring, the substituent being selected fromthe group of lower alkyl, lower alkoxy and halo, or L represents thesame or different of two substituents on the aromatic ring, thesubstituents being selected from the group of lower alkyl, lower alkoxyand halo, provided that any two substituents do not interfere with eachothers presence;

R² is --N(R⁸)CH(R⁹)C(O)-- wherein R⁸ is hydrogen or lower alkyl and R⁹is hydrogen, lower alkyl, lower alkyl monosubstituted with hydroxy,methoxy, methylthio or benzyloxy, lower cycloalkyl, (lowercycloalkyl)methyl, benzyl, 4-imidazolylmethyl, 2-thienylmethyl,2-thiazolylmethyl, (4-hydroxyphenyl)methyl, [4-(loweralkoxy)phenyl]methyl, --CH(OH)C₆ H₅, --(CH₂)₄ --NH₂, or--(CH₂)_(n)C(O)OR¹⁰ or --(CH₂)_(n) C(O)NR¹¹ R¹² wherein n is the integer one, twoor three, R¹⁰ is hydrogen, a straight or branched chain alkyl containingone to ten carbon atoms, or phenyl(lower)alkyl and R¹¹ and R¹² eachindependently is hydrogen, lower alkyl, phenyl(lower)alkyl, or R¹¹ andR¹² together with the nitrogen to which they are joined form apyrrolidino, piperidino, morpholino, piperazino or 4-(loweralkyl)piperazino;

Y is a derived amino acid radical of the formula --NH--CH(R¹³)--W--wherein R¹³ is lower alkyl, (lower cycloalkyl)methyl, --CH₂ CH₂ SCH₃,benzyl or benzyl substituted on the aromatic portion thereof withhydroxy or lower alkoxy, and W is --CH(OH)CH₂ C(O)-- or --CH₂NHCH(R¹⁴)C(O)-- wherein R¹⁴ has the same meaning as defined for R¹³ ;and

R³ is absent or is --N(R¹⁵)CH(R¹⁶)C(O)-- wherein R¹⁵ is hydrogen orlower alkyl and R¹⁶ has the same meaning as defined herein for R⁹ ; and

R⁴ is [--NR¹⁷ CH(R¹⁸)C(O)]_(p) --Z wherein R¹⁷ is hydrogen or loweralkyl, R¹⁸ has the same meaning as defined herein for R⁹, p is theinteger zero or one, and Z is hydrogen, lower alkoxy, benzyloxy or--NR¹⁹ R²⁰ wherein R¹⁹ and R²⁰ each independently is hydrogen, loweralkyl or phenyl(lower)alkyl, or R¹⁹ and R²⁰ together with the nitrogenatom to which they are joined form a pyrrolidino, piperidino,morpholino, piperazino or 4-(lower alkyl)piperazino; or

R⁴ is --NR¹⁷ CR¹⁸ (R²¹)CH₂ OH wherein R¹⁷ and R¹⁸ are as defined hereinand R²¹ is hydrogen, lower alkyl or lower alkyl monosubstituted withhydroxy; or a therapeutically acceptable salt thereof.

A preferred group of the peptides of this invention for inhibiting HIVprotease is represented by formula 1 wherein R¹ is the radical offormula 2 wherein R⁵ hydrogen, lower alkyl, or R⁷ OC(O)-- or R⁷ C(O)--wherein R⁷ is lower alkyl, R⁶ is hydrogen, lower alkyl or benzyl, L ishydrogen or one or two halo substituents as defined herein; R² is--N(R⁸)CH(R⁹)CO-- wherein R⁸ is hydrogen or methyl and R⁹ is loweralkyl, lower cycloalkyl, cyclopropylmethyl, cyclohexylmethyl, --CH₂ CH₂COOH, --CH₂ CH₂ CONH₂, --CH₂ CONH₂ or benzyl; Y is --NHCH(R¹³)--W--wherein R¹³ is lower alkyl, (lower cycloalkyl)methyl, --CH₂ CH₂ --SCH₃,benzyl or (4-methoxyphenyl)methyl and W is --CH(OH)--CH₂ C(O)-- or --CH₂NHCH(R¹⁴)C(O)-- wherein R¹⁴ is lower alkyl or (lower cycloalkyl)methyl;R³ is --N(R¹⁵)CH(R¹⁶)C(O)-- wherein R¹⁵ is hydrogen or methyl and R¹⁶ ishydrogen, lower alkyl, cyclopropylmethyl, cyclohexylmethyl, benzyl,--CH(OH)--CH₃ or --(CH₂)_(n) C(O)OR¹⁰ or --(CH₂)_(n) C(O)NR¹¹ R¹²wherein n is the integer one, two or three, R¹⁰ is hydrogen or astraight or branched chain alkyl containing one to ten carbon atoms, andR¹¹ and R¹² each independently is hydrogen, methyl or ethyl; and R⁴either is --N(R¹⁷)CH(R¹⁸)C(O)--Z wherein R¹⁷ is hydrogen or methyl andR¹⁸ is lower alkyl, lower alkyl monosubstituted with a hydroxy or abenzyloxy, cyclopropylmethyl, cyclohexylmethyl, --CH(OH)C₆ H₅ or benzyl,and Z is hydroxy or NR¹⁹ R²⁰ wherein R¹⁹ and R²⁰ each independently ishydrogen, methyl, ethyl or 2-methylbutyl, or R¹⁹ and R²⁰ together withthe nitrogen atom to which they are attached form a pyrrolidino orpiperidino; or R⁴ is --NR¹⁷ CR¹⁸ (R²¹)CH₂ OH wherein R.sup. 17 and R¹⁸are as defined in the last instance and R²¹ is hydrogen, lower alkyl orlower alkyl substituted with a hydroxy; or a therapeutically acceptablesalt thereof.

Using the conventional three letter system for designating amino acidresidues (see the second paragraph of the Details of the Invention,below), a more preferred group of the peptides is represented by formula1 wherein R¹ is the radical of formula 2 wherein R⁵ is hydrogen ortertiary-butyloxycarbonyl, R⁶ is hydrogen, methyl, ethyl or benzyl, andL is hydrogen or one or two halo substituents at position 3 or positions3 and 5, respectively, of the aromatic ring; R² is Val, Ala, Leu, Ile,Gly, Tbg, Cpa, Cha, Glu, Gln, Asn or Phe; Y is --NHCH(R¹³)--W-- whereinR¹³ is 1-methylethyl, Glu, Gln, 2-methylpropyl, cyclohexylmethyl, --CH₂CH₂ SCH₃, benzyl or (4-methoxyphenyl)methyl, and W is --CH(OH)CH₂ C(O)--or --CH₂ NHCH(R¹⁴)C(O)-- wherein R¹⁴ is 2-methylpropyl orcyclohexylmethyl; R³ is absent or is Leu, Nle, Ile, Val, Ala, Gly, Cha,Phe, Thr, Glu, Gln, Asp or Asn; R⁴ is Leu-NH₂ , Leu-OH, Ile-NH₂, Ile-OH,Val-NH₂, Val-OH, Ala-NH₂, Ala-OH, Thr(OBzl)-NH₂, Cpa-NH₂, Cpa-OH,Cha-NH₂, Cha-OH, Phe-NH₂, Phe-NH[CH₂ CH(CH₃)C₂ H₅ ], Phe-N(C₂ H₅)₂ orPhe-OH, or R⁴ is --NHCR¹⁸ (R²¹)CH₂ OH wherein R¹⁸ is lower alkyl, --CH₂OH, --CH(OH)CH₃, --CH₂ CH₂ OH or --CH(OH)--C₆ H₅ and R²¹ is hydrogen,methyl or --CH₂ OH; or a therapeutically acceptable salt thereof.

A most preferred group for inhibiting HIV protease is represented bycompounds of formula 1 in which R¹ is the radical of formula 2 whereinR⁵ is hydrogen or tertiary-butyloxycarbonyl, R⁶ is hydrogen or benzyland L is hydrogen or one or two halo substituents at position 3 orpositions 3 and 5, respectively, of the aromatic ring; R² and Y are asdefined in the last instance; R³ is Leu, Nle, Ile, Val, Ala, Cha, Glu orGln; and R⁴ either is Ile-NH₂, Ala-NH₂, Thr(OBzl)-NH₂, Cha-NH₂ orPhe-NH₂, or is --NHCR¹³ (R²¹)CH₂ OH wherein R¹³ is 1-methylethyl,1-methylpropyl, 2-methylpropyl, --CH₂ OH or --CH(OH)C₆ H₅ and R²¹ ishydrogen or methyl; or a therapeutically acceptable salt thereof.

Included within the scope of this invention is a pharmaceuticalcomposition for treating HIV infections in a human, comprising acompound of formula 1, or a therapeutically acceptable salt thereof, anda pharmaceutically acceptable carrier.

The scope of the invention includes as well a method for treating HIVinfections in a human comprising administering thereto an effectiveamount of the compound of formula 1, or a therapeutically acceptablesalt thereof.

Also included within the scope is a method for protecting human cellsagainst HIV pathogenesis comprising treating said cells with an anti-HIVeffective amount of a compound of formula 1, or a therapeuticallyacceptable salt thereof.

Furthermore, the scope of the invention includes pharmaceuticalformulations and a method of treatment for renin-associated hypertensionor a method of treating congestive heart failure in a mammal wherein theactive agent is a peptide of formula 1. The method comprisesadministering to the mammal a renin-lowering effective amount of thepeptide of formula 1 or a therapeutically acceptable salt thereof.

Processes for preparing the compounds of formula 1 are describedhereinafter.

DETAILS OF THE INVENTION GENERAL

The term "residue" with reference to an amino acid means a radicalderived from the corresponding α-amino acid by eliminating the hydroxylof the carboxy group and one hydrogen of the α-amino group.

In general, the abbreviations used herein for designating the aminoacids and the protective groups are based on recommendations of theIUPAC-IUB Commission of Biochemical Nomenclature, see European Journalof Biochemistry, 138, 9 (1984). For instance, Val, Glu, Gln, Ala, Ile,Asp, Phe, Leu, Asn and Gly represent the residues of L-valine,L-glutamic acid, L-glutamine, L-alanine, L-isoleucine, L-aspartic acid,L-phenylalanine, L-leucine, L-asparagine and glycine, respectively. Thesymbols "Cpa" and "Cha" represent the residues of2(S)-amino-3-cyclopropylpropionic acid (L-cyclopropylalanine) and2(S)-amino-3-cyclohexylpropionic acid (L-cyclohexylalanine),respectively. The symbols "Nle" and "Tbg" represent the residues of2(S)-aminohexanoic acid (L-norleucine) and2(S)-amino-3,3-dimethylbutyric acid, respectively.

The term "lower alkyl" as used herein, either alone or in combinationwith a radical, means straight chain alkyl radicals containing one tofour carbon atoms and branched chain alkyl radicals containing three tofour carbon atoms and includes methyl, ethyl, propyl, butyl,1-methylethyl, 1-methylpropyl, 2-methylpropyl and 1,1-dimethylethyl.

The term "lower cycloalkyl" as used herein, either alone or incombination with a radical, means saturated cyclic hydrocarbon radicalscontaining from three to six carbon atoms and includes cyclopropyl,cyclobutyl, cyclopentyl and cyclohexyl.

The term "lower alkoxy" as used herein means straight chain alkoxyradicals containing one to four carbon atoms and branched chain alkoxyradicals containing three to four carbon atoms and includes methoxy,ethoxy, propoxy, 1-methylethoxy, butoxy and 1,1-dimethylethoxy. Thelatter radical is known commonly as tertiary-butyloxy.

The symbol "Boc" represents 1,1-dimethylethoxycarbonyl, known commonlyas tertiary-butyloxycarbonyl. The symbol "C₆ H₅ " represents a phenylradical.

The term "halo" as used herein means a halo radical selected from bromo,chloro, fluoro or iodo.

With reference to Y of general formula 1, the radical "--NHCH(R¹³)--W--"wherein R¹³ is as defined hereinabove and W is --CH(OH)CH₂ CO--represents the radical derived from the δ-amino acid known as statine(i.e. 4(S)-amino-3(S)-hydroxy-6-methylheptanoic acid) and its closeanalogs. The radical is derived by eliminating the hydroxyl of thecarboxy group and one hydrogen of the amino group of the correspondingδ-amino acid. Each such radical has two chiral centers and thus canexist in various optically active or optically inactive forms. All formsare included for the peptides of formula 1 and for the appropriateintermediates therefore, the 4(S)-amino-3(S)-hydroxy enantiomers beingpreferred. The requisite 4-amino-3-hydroxy pentanoic acids for preparingthe synthon to incorporate the radical into the peptide of formula 1 canbe prepared by methods described by D. H. Rich and E. T. O. Sun, J. Med.Chem., 23, 27 (1980), and references therein.

The term "Sta" represents the radical --NHCH(2-methylpropyl)CH(OH)CH₂C(O)--, derived from statine. The term "ACHPA" represents the radical--NHCH(cyclohexylmethyl)CH(OH)CH₂ C(O)--, derived from4-amino-5-cyclohexyl-3-hydroxy-pentanoic acid, and the term "AHPPA"represents the radical --NHCH(benzyl)CH(OH)CH₂ C(O)--, derived from4-amino-3-hydroxy-5-phenylpentanoic acid. The 4(S)-amino-3(S)-hydroxyenantiomers of these last three embodiments are preferred. Unlessdesignated otherwise by an antecedent such as (3R, 4R), the terms Sta,ACHPA and AHPPA represent their respective 4(S)-amino-3(S)-hydroxyenantiomers.

The asymmetric carbon atoms of the R¹, R², R³ and R⁴ units of thepeptides of formula 1 have an S configuration, except those residing inthe side chain of the amino acid or derived amino acid residues whichmay also have the R configuration.

The term "amino" as used herein means an amino radical of formula --NH₂.The term "lower alkylamino" as used herein means alkylamino radicalscontaining one to six carbon atoms and includes methylamino, ethylamino,propylamino, 1-methylethylamino and 2-methylbutylamino. The term"di(lower alkyl)amino" means an amino radical having two lower alkylsubstituents each of which contains one to six carbon atoms and includesdimethylamino, diethylamino, ethylmethylamino and the like.

Additional abbreviations or symbols used hereafter for derived aminoacid residues include Boc-Tyr(O-AcOH) for the R¹ radical N-Boc-O⁴-(carboxymethyl)-L-tyrosyl and Boc-Tyr(O-AcOBz) for the R¹ radicalN-Boc-O⁴ -(benzyloxycarbonylmethyl)tyrosyl. Also note that when Y is theradical --NHCH--(R¹³)--W-- wherein R¹³ is as defined hereinabove and Wis --CH₂ NHCH(R¹⁴)CO-- wherein R¹⁴ is as defined hereinabove, theradical is equivalent to two adjoining, corresponding amino acidresidues wherein the amide bond joining the two residues is reduced.According to convention, the latter radical can be expressedsymbolically as two amino acid residues (in the three letter system)with the symbol "ψ[CH₂ NH]" inserted between the designation of the twoadjoining amino acid residues. Accordingly, for example, the peptide offormula 1 wherein R¹ is N--Boc--O⁴ -(carboxymethyl)-L-tyrosyl, R² isAla, Y is --NHCH(benzyl)CH₂ NHCH(2-methylpropyl) with two (S)-asymmetriccenters, R³ is Ala, R⁴ is Phe-NH₂ is designated asBoc-Tyr(O-AcOH)Ala-Pheψ[CH₂ NH]Leu-Ala-Phe-NH₂.

The term "coupling agent" as used herein means an agent capable ofeffecting the dehydrative coupling of an amino acid or peptide freecarboxy group with a free amino group of another amino acid or peptideto form an amide bond between the reactants. The agents promote orfacilitate the dehydrative coupling by activating the carboxy group.Descriptions of such coupling agents and activated groups are includedin general textbooks of peptide chemistry; for instance, E. Schroder andK. L. Lubke, "The Peptides", Vol. 1, Academic Press, New York, N.Y.,1965, pp 2-128, and K. D. Kopple, "Peptides and Amino acids", W. A.Benjamin, Inc., New York, N.Y., 1966, pp 33-51. Examples of couplingagents are thionyl chloride, diphenylphosphoryl azide,dicyclohexylcarbodiimide, N-hydroxysuccinimide, or1-hydroxybenzotriazole in the presence of dicyclohexylcarbodiimide. Avery practical and useful coupling agent is(benzotriazol-1-yloxy)tris(dimethylamino)phosphoniumhexafluorophosphate, described by B. Castro et al., Tetrahedron Letters,1219 (1975), see also D. Hudson, J. Org. Chem., 53, 617 (1988), eitherby itself or in the presence of 1-hydroxybenzotriazole.

The term "pharmaceutically acceptable carrier" as used herein means anon-toxic, generally inert vehicle for the active ingredient, which doesnot adversely affect the ingredient.

The term "effective amount" as used herein means a pre-determined amountof the peptide of this invention sufficient to be effective against HIVin vivo or renin-associated hypertension.

PROCESS

The peptides of formula 1 can be prepared by processes which incorporatetherein methods commonly used in peptide synthesis such as classicalsolution coupling of amino acid residues and/or peptide fragments, andif desired solid phase techniques. Such methods are described, forexample, by E. Schroder and K. Lubke, cited above, in the textbookseries, "The Peptides: Analysis, Synthesis, Biology", E. Gross et al.,Eds., Academic Press, New York, N.Y., 1979-1987, Volumes 1 to 8, and byJ. M. Stewart and J. D. Young in "Solid Phase Peptide Synthesis", 2nded., Pierce Chem. Co., Rockford, IL, USA, 1984.

A common feature of the aforementioned processes for the peptides is theprotection of the labile side chain groups of the various amino acidresidues or derived amino acid residues with suitable protective groupswhich will prevent a chemical reaction from occurring at that site untilthe protective group is ultimately removed. Usually also common is theprotection of an α-amino group on an amino acid or a fragment while thatentity reacts at the carboxy group, followed by the selective removal ofthe α-amino protective group to allow subsequent reaction to take placeat that location. Usually another common feature is the initialprotection of the C-terminal carboxyl of the amino acid residue orpeptide fragment, if present, which is to become the C-terminal functionof the peptide, with a suitable protective group which will prevent achemical reaction from occurring at that site until the protective groupis removed after the desired sequence of the peptide has been assembled.

Another feature of the preparation of the peptides of formula 1 is theincorporation into the peptide of the unit Y. As noted previously theunit Y can be either of two subunits, i.e. --NHCH(R¹³)CH(OH)CH₂ C(O)--wherein R¹³ is as defined herein or --NHCH(R¹³)CH₂ NHCH(R¹⁴)C(O)--wherein R¹³ and R¹⁴ are as defined herein. The first-mentioned subunitcan readily be incorporated into the peptide by coupling the N-protectedderivative of the corresponding 4-amino-3-hydroxypentanoic acid (offormula NH₂ --CH(R¹³)CH(OH)CH₂ C(O)OH), noted above, at the appropriatepoint during the preparation of the peptide by the classical methods ofcoupling of amino acid residues or fragments. The second-mentionedsubunit, --NHCH(R¹³)CH₂ NH--CH(R¹⁴)C(O)-- can be incor-porated byforming the linear peptidyl framework of the peptide of formula 1, or afragment thereof, by a reductive alkylation between two sub-fragments,each sub-fragment containing a precursor portion of the Y unit and atleast one of the sub-fragments containing one or more of the amino acidunits, whereby the CH₂ NH bond of the Y unit is formed; for example, thereductive N-alkylation of the dipeptide of formula NH₂ CH(R¹⁴)C(O)--R³--R⁴ with Boc-NHCH(R¹³)--CHO in the presence of sodium cyanoborohydrideto give the fragment Boc-NHCH(R¹³)CH₂ NHCH(R¹⁴)C(O)--R³ --R⁴.

With reference to the process products, the peptides of formula 1 can beprepared by:

(i) coupling the derived amino acid of formula 2a ##STR2## whereinR^(5A) is lower alkyl or R⁷ OC(O)-- or R⁷ C(O)-- wherein R⁷ is asdefined hereinabove, R^(6A) is lower alkyl, lower cycloalkyl, (lowercycloalkyl)methyl, phenyl, phenyl monosubstituted with lower alkyl,lower alkoxy or halo, benzyl or benzyl monosubstituted with lower alkyl,lower alkoxy or halo, and L is as defined herein; with a fragment offormula H--R^(2A) --Y--R^(3A) --R^(4A) wherein R^(2A), R^(3A) and R^(4A)have the same meaning as defined herein for R², R³ and R⁴ respectively,except that side chain amino and carboxy groups, and a C-terminalcarboxy group, if present, are replaced with corresponding protectedgroups and Y is as defined herein; followed, if required, by aminodeprotection and/or carboxy deprotection, to give the correspondingpeptide of formula 1. Note that benzyl or substituted benzyl of R^(6A)can serve a dual role, i.e. serve as the progenitor for thecorresponding radical in the ultimate product of the synthesis or serveas a carboxy protecting group. When required, such carboxy protectinggroups can be selectively removed by known methods (e.g. hydrogenation)in the presence of other carboxy protecting groups (e.g. classical acidsensitive protecting groups) in the pentultimate or next to thepentultimate intermediate of the process. Also note that the radicals R⁷OC(O)-- and R⁷ C(O)-- can serve a dual role as a progenitor for thecorresponding radical in the final product or serve as an aminoprotecting group.

Alternatively, the peptides of formula 1 in which R¹ is the derivedamino acid radical of formula 2 in which R⁵, R⁶ and L are as definedherein, and R², Y, R³ and R⁴ are as defined herein can be prepared bysubjecting the intermediate of formula 3 ##STR3## wherein R^(5A), L,R^(2A), Y, R^(3A) and R^(4A) are as defined hereinabove, to O-alkylationwith an alkylating agent of formula XCH₂ C--(O)OR^(6A) wherein X isbromo, chloro or iodo and R^(6A) is as defined hereinabove, in thepresence of a suitable strong base; followed, if required, bydeprotection to obtain the desired corresponding peptide of formula 1.

The intermediate of formula 3 can be prepared by conventional methods;for example, see D. F. Veber et al., European patent application,77,028, published Apr. 20, 1983. Suitable strong bases for theabove-noted alkylation include alkali metal carbonates, preferablypotassium carbonate; alkali metal hydroxides, preferably sodiumhydroxide or potassium hydroxide; or alkali metal hydrides, preferablysodium hydride.

The peptide of formula 1 of this invention can be obtained in the formof a therapeutically acceptable salt.

In the instance where a particular peptide has a residue which functionsas a base, examples of such salts are those with organic acids, e.g.acetic, lactic, succinic, benzoic, salicylic, methanesulfonic orp-toluenesulfonic acid, as well as polymeric acids such as tannic acidor carboxymethyl cellulose, and also salts with inorganic acids such ashydrohalic acids, e.g. hydrochloric acid, or sulfuric acid, orphosphoric acid. If desired, a particular acid addition salt isconverted into another acid addition salt, such as a non-toxic,pharmaceutically acceptable salt, by treatment with the appropriate ionexchange resin in the manner described by R. A. Boissonnas et al., Helv.Chim. Acta, 43, 1849 (1960).

In the instance where a particular peptide has one or more free carboxygroups, examples of such salts are those with the sodium, potassium orcalcium cations, or with strong organic bases, for example,triethylamine or N-methylmorpholine.

In general, the therapeutically acceptable salts of the peptides offormula 1 are biologically fully equivalent to the peptides themselves.

BIOLOGICAL ASPECTS

The HIV protease inhibiting properties and the cell protective effectagainst HIV pathogenesis of the peptides of formula 1, or atherapeutically acceptable salt thereof, can be demonstrated bybiochemical, microbiological and biological procedures.

A particular useful procedure for demonstrating the HIV proteaseinhibiting properties of the peptides of formula 1 or theirtherapeutically acceptable salts is the "Recombinant HIV Protease HLPCAssay". The procedure is based on the capacity of the test compound toinhibit enzymatic cleavage by HIV protease of a decapeptide (thesubstrate) having an amino acid sequence which includes a known HIVprotease cleavage site of the HIV polyprotein; see H. G. Krausslich etal., Proc. Natl. Acad. Sci. USA, 86, 807 (1989). Details of this assaytogether with the results obtained for exemplified peptides of formula 1are described in the examples hereinafter.

The cell protective effect of the peptides or their therapeuticallyacceptable salts can be demonstrated by microbiological procedures forevaluating the effect of test compounds in inhibiting thecytopathogenicity of HIV in human T4 cell lines; for example, see M.Baba et al., Biochem, Biophys. Res. Comm., 142, 128 (1987).

When a peptide of this invention, or a therapeutically acceptable saltthereof, is used to combat HIV infections in a human, the peptide can beadministered orally, topically or parenterally, in a vehicle comprisingone or more pharmaceutically acceptable carriers, the proportion ofwhich is determined by the solubility and chemical nature of thepeptide, chosen route of administration and standard biologicalpractice. For oral administration, the peptide or a therapeuticallyacceptable salt thereof can be formulated in unit dosage forms such ascapsules or tablets each containing a predetermined amount of the activeingredient, ranging from about 25 to 500 mg, in a pharmaceuticallyacceptable carrier. For topical administration, the peptide can beformulated in a pharmaceutically acceptable vehicle containing 0.1 to 10percent, preferably 0.5 to 5 percent, of the active agent. Suchformulations can be in the form of a cream, lotion, sublingual tablet,or preferably a transdermal patch or buccal patch.

For parenteral administration, the peptide of formula 1 is administeredby either intravenous, subcutaneous or intramuscular injection, incompositions with pharmaceutically acceptable vehicles or carriers. Foradministration by injection, it is preferred to use the peptide insolution in a sterile aqueous vehicle which may also contain othersolutes such as buffers or preservatives as well as sufficientquantities of pharmaceutically acceptable salts or of glucose to makethe solution isotonic.

Suitable vehicles or carriers for the above noted formulations can befound in standard pharmaceutical texts, e.g. in "Remington'sPharmaceutical Sciences", 16th ed, Mack Publishing Company, Easton,Penn., 1980.

The dosage of the peptide will vary with the form of administration andthe particular active agent chosen. Furthermore, it will vary with theparticular host under treatment. Generally, treatment is initiated withsmall dosages substantially less than the optimum dose of the peptide.Thereafter, the dosage is increased by small increments until theoptimum effect under the circumstances is reached. In general, thepeptide is most desirably administered at a concentration level thatwill generally afford antivirally effective results without causing anyharmful or deleterious side effects.

For oral adminstration, the peptide or a therapeutically acceptable saltis administered in the range of 1.0 to 75 mg per kilogram of body weightper day, with a preferred range of 2.5 to 20 mg per kilogram.

With reference to systemic adminstration, the peptide of formula 1 isadministered at a dosage of 10 mcg to 1000 mcg per kilogram of bodyweight per day, although the aforementioned variations will occur.However, a dosage level that is in the range of from about 50 mcg to 500mcg per kilogram of body weight per day is most desirably employed inorder to achieve effective results.

Although the formulations disclosed hereinabove are effective andrelatively safe medications for treating HIV infections, the possibleconcurrent administration of these formulations with other antiviralmedications or agents to obtain beneficial results is not excluded. Suchother antiviral medications or agents include soluble CD4, zidovudine,dideoxycytidine, phosphonoformate, ribavarin, antiviral interferons(e.g. α-interferon or interleukin-2) or aerosol pentamidine.

The peptides of formula 1 also possess the ability to inhibit reninactivity. The renin inhibiting activity of the compounds can bedemonstrated in standard pharmacological tests such as those describedby M. G. Bock et al., J. Med. Chem., 31, 1918 (1988). As such thepeptides are indicated for the diagnosis, prophylaxis and treatment ofrenin-associated hypertension and for the treatment of congestive heartfailure in mammals including humans. For the latter purposes orindication, the peptides can be formulated and administered in the samemanner as described above, but usually at higher dosages which can bedetermined conventionally by using well known pharmacological protocols.

A preferred group of the peptides for inhibiting renin is represented byformula 1 wherein R¹ is the radical of formula 2 wherein R⁵ is R⁷OC(O)-- wherein R⁷ is lower alkyl, R⁶ is hydrogen or benzyl and L ishydrogen; R² is --N(R⁸)CH(R⁹)CO-- wherein R⁸ is hydrogen and R⁹ is loweralkyl, lower alkyl monosubstituted with hydroxy, benzyl,4-imidazolymethyl, 2-thienylmethyl or 2-thiazolymethyl, Y is Sta, ACHPAor AHPPA, R³ is absent or is --N(R¹⁵)CH(R¹⁶)CO-- wherein R¹⁵ is hydrogenand R¹⁶ is lower alkyl, and R⁴ is either --NR¹⁷ CH(R¹⁸)--C(O)--Z whereinR¹⁷ is hydrogen, R¹⁸ is lower alkyl, lower alkyl monosubstituted withhydroxy or benzyloxy, --CH(OH)C₆ H₅ or benzyl, and Z is hydrogen oramino, or R⁴ is --NR¹⁷ CR¹⁸ (R²¹)--CH₂ OH wherein R¹⁷ is hydrogen, R¹⁸is lower alkyl, lower alkyl monosubstituted with a hydroxy, --CH(OH)C₆H₅ or benzyl and R²¹ is hydrogen or lower alkyl, or a therapeuticallyacceptable salt thereof.

A more preferred group of peptides for inhibiting renin is representedby formula 1 wherein R¹ is the radical of formula 2 wherein R⁵ istertiary-butyloxycarbonyl, R⁶ is hydrogen, methyl or benzyl and L ishydrogen, R² is Val, Ala or Phe, Y is as defined in the last instance,R³ is absent or Ala, Ile or Leu, and R⁴ is Ala-NH₂, Phe-NH₂ or --NHCR¹⁸(R²¹)CH₂ OH wherein R¹⁸ is --CH₂ OH, --CH(OH)CH₃ or --CH(OH)C₆ H₅ andR²¹ is hydrogen or methyl, or a therapeutically acceptable salt thereof.

The following examples illustrate further this invention. Solutionpercentages or ratios express volume to volume relationship, unlessstated otherwise. Abbreviations used in the examples include Boc:t-butyloxycarbonyl; BOP:(benzotriazol1-yloxy)tris(dimethylamino)-phosphoniumhexafluorophosphate; Bzl: benzyl; DMF: dimethyl formamide; Et₂ O:diethyl ether; Fm: 9-fluorenylmethyl; HPLC: high performance liquidchromatography: MeOH: methanol; TFA: trifluoroacetic acid; THF:tetrahydrofuran.

EXAMPLE 1 Preparation of N-[N-[N-[N-[N-Boc-O⁴-(Benzyloxycarbonylmethyl)-L-tyrosyl]-L-valyl]-4(S)-amino-3(S)-hydroxy-5-cyclohexylpentanoyl]-L-leucyl]-L-phenylalaninamide(Boc-Tyr(OAcOBzl)-Val-ACHPA-Leu-Phe-NH₂.

a) Boc-Phe-NH₂ : Isobutyl chloroformate (2.86 mL, 22 mmol) was addeddropwise at 0° C. to a stirred solution of N-methylmorpholine (2.42 mL,22 mmol) and Boc-Phe-OH (5.31 g, 20 mmol) in dry THF. The resultingsolution was stirred for an additional 30 min at 0° C. Thereafter, a 28%aqueous solution of ammonia (5 mL) was added dropwise over 5 min. Thesolvent was evaporated and the residue was dissolved in ethyl acetate.The solution was washed successively with a 5% aqueous solution ofcitric acid (three times), saturated aqueous NaHCO₃ (three times) andsaturated aqueous NaCl. The organic solution was dried over Na₂ SO₄ andevaporated to afford Boc-Phe-NH₂ (5.0 g, 94%) as a white solid.

b) Boc-Leu-Phe-NH₂ : A solution of Boc-Phe-NH₂ (6.46 g, 24.4 mmol) in 6NHCl/dioxane (84 mL) was stirred at room temperature (20°-22° C.) under anitrogen atmosphere for 30 min. The solvent was evaporated and theresidue was dried under high vacuum. The solid residue was suspended indry CH₃ CN (300 mL) and the resulting mixture was cooled to 0°-5° C. andstirred under a nitrogen atmosphere. Dry Et₃ N (3.7 mL, 26 mmol) wasadded, followed by Boc-Leu-OH(monohydrate) (5.4 g, 22 mmol), BOP (10.69g, 24.2 mmol) and more Et₃ N (7.4 mL, 53 mmol). After 1.5 h, more BOP(4.28 g, 9.7 mmol) and Et₃ N (1.48 mL, 10.6 mmol) were added. Thereaction was stirred for an additional 15 min, then the CH₃ CN wasevaporated under reduced pressure and the residue was partitionedbetween a saturated aqueous solution of NaCl (200 mL) and ethyl acetate(3 times 200 mL). The combined organic solutions were washedsuccessively with 100 mL each of a 10% aqueous solution of citric acid,water, a 5% aqueous solution of NaHCO₃ (three times) and water. Theorganic solution was dried over MgSO₄ and concentrated under reducedpressure. Chromatography of the residue over silica gel (eluent=ethylacetate) gave a white gum which was triturated with Et₂ O/hexane. Theresulting solid was collected to afford Boc-Leu-Phe-NH₂ (7.7 g, 93%) asa white solid. Mass spectrum: 378 (M+H)⁺. Amino acid analysis: Leu, 100;Phe, 1.00.

c) Boc-ACHPA-Leu-Phe-NH₂ : A solution of Boc-Leu-Phe-NH₂ (100 mg, 0.26mmol) in 6N HCl/dioxane (1 mL) was stirred at room temperature under anitrogen atmosphere for 15 min. The solvent was evaporated and theresidue was dried under high vacuum for 1 h. The solid was suspended indry CH₃ CN (2 mL) and stirred under a nitrogen atmosphere. The solutionwas adjusted to pH 8 (wet pH paper) by the addition ofN-methylmorpholine, then Boc-ACHPA-OH (83 mg, 0.26 mmol) was added. Thesolution was stirred at room temperature for 1 h (during which time pH 8was maintained by the occasional addition of N-methylmorpholine). Themixture was poured into a saturated aqueous solution of NaCl. Theaqueous solution was extracted twice with ethyl acetate. The combinedorganic extracts were washed successively with ice-cold 0.5N aqueousHCl, 10% aqueous Na₂ CO₃ (twice) and saturated aqueous NaCl (threetimes). The organic solution was dried over Na₂ SO₄ and the solvent wasevaporated. Chromatography of the residue over silica gel, eluting with5% methanol in chloroform, afforded Boc-ACHPA-Leu-Phe-NH₂ (138 mg, 92%)as a white solid.

d) Boc-Val-ACHPA-Leu-Phe-NH₂ : A solution of Boc-ACHPA-Leu-Phe-NH₂ (72mg, 0.125 mmol) in 6 NHCl/dioxane (1 mL) was stirred at room temperatureunder a nitrogen atmosphere for 15 min. The solvent was evaporated andthe residue was dried under high vacuum for 1 h. The solid was suspendedin dry CH₃ CN (2 mL) and stirred under a nitrogen atmosphere. Thesolution was adjusted to pH 8 (wet pH paper) by the addition ofN-methylmorpholine, then Boc-Val-OH (27 mg, 0.13 mmol) was added, the pHwas again adjusted to pH 8 (as before) and BOP (55 mg, 0.13 mmol) wasadded. The solution was stirred at room temperature for 1 h (duringwhich time pH 8 was maintained by the occasional addition ofN-methylmorpholine). The mixture was poured into a saturated aqueoussolution of NaCl. The aqueous solution was extracted twice with ethylacetate. The combined organic extracts were washed successively withice-cold 0.5N aqueous HCl, 10% aqueous Na₂ CO₃ (twice) and saturatedaqueous NaCl (three times). The organic solution was dried over Na₂ SO₄and the solvent was evaporated. Chromatography of the residue oversilica gel, eluting with 5% methanol in chloroform, affordedBoc-Val-ACHPA-Leu-Phe-NH₂ (71 mg, 84%) as a white solid.

e) Boc-Tyr-Val-ACHPA-Leu-Phe-NH₂ : A solution ofBoc-Val-ACHPA-Leu-Phe-NH₂ (175 mg, 0.26 mmol) in 6N HCl/dioxane (1 mL)was stirred at room temperature under a nitrogen atmosphere for 15 min.The solvent was evaporated and the solid residue was dried under highvacuum for 1 h. The solid was suspended in dry CH₃ CN (3 mL). Themixture was stirred under a nitrogen atmosphere. The solution wasadjusted to pH 8 (wet pH paper) by the addition of N-methylmorpholine,then Boc-Tyr-OH (73 mg, 0.26 mmol) was added, the pH was again adjustedto pH 8 (as before) and BOP (115 mg, 0.26 mmol) was added. The solutionwas stirred at room temperature for 90 min (during which time pH 8 wasmaintained by the occasional addition of N-methylmorpholine). Themixture, which became a gel, was mixed with ethyl acetate. The mixturewas sonicated to give a homogenous suspension. The solid in thesuspension was collected on a filter, washed with ethyl acetate anddissolved in 10% methanol in chloroform. The solution was filteredthrough silica gel to afford Boc-Tyr-Val-ACHPA-Leu-Phe-NH₂ (144 mg, 66%)as a white solid upon evaporation of the eluent. Mass spectrum: 837(M+H)⁺.

The latter compound (130 mg, 0.155 mmol) was mixed with K₂ CO₃ (86 mg,0.62 mmol) in DMF (1 mL). Benzyl bromoacetate (37 μL, 0.230 mmol) wasadded to the stirred mixture. The resulting mixture was stirred at roomtemperature under an atmosphere of nitrogen for 14 h. The mixture waspoured into water (50 mL) and the resulting suspension stirredvigorously for 5 min. The precipitated solid was collected on a filterand washed successively with portions of ethyl acetate and Et₂ O. Thesolid was redissolved in DMSO and precipited by the addition of water.The solid was collected by filtration and dried under high vacuum toafford the title compound (110 mg, 72%) as a white solid. Mass spectrum:985 (M+H)⁺. Amino acid analysis: Leu, 1.03; Phe, 0.94; Val, 1.03; ACHPA,0.96; Tyr(O-AcOH), 0.79.

EXAMPLE 2 H-Tyr(O-AcOBzl)-Val-ACHPA-Leu-Phe-NH₂.HCl

A solution of Boc-Tyr(O-AcOBzl)-Val-ACHPA-Leu-Phe-NH₂ (20 mg, 0.020mmol) in 6N HCl/dioxane (1.5 mL) was stirred at room temperature under anitrogen atmosphere for 20 min. The solvent was evaporated and theresidue was dried under oil pump vacuum for 1 h. The solid wastriturated with Et₂ O and the resulting suspension was filtered. Theprecipitate was collected and dried under high vacuum for 17 h at roomtemperature to afford the title compound (12 mg, 65%) as a white solid.Mass spectrum: 886 (M-Cl)⁺. Amino acid analysis: Leu, 1.03; Phe, 0.93;Val, 1.04; ACHPA, 0.91; Tyr(OAcOH), 0.80.

EXAMPLE 3 Boc-Tyr(O-AcOH)-Val-ACHPA-Leu-Phe-NH₂

MeOH (16 mL) was added to a solution ofBoc-Tyr(O-AcOBzl)-Val-ACHPA-Leu-Phe-NH₂ (74 mg, 0.075 mmol) in DMF (4mL). Under an atmosphere of argon, 10% palladium on carbon (7.4 mg) wasadded. The mixture was shaken on a Parr apparatus under an atmosphere ofH₂ (at 45 psi) for 2 h. The mixture was filtered through a 45 μmmembrane and the filtrate was evaporated. The residue was dried underhigh vacuum to afford the title compound (47 mg, 70%). Mass spectrum:917 (M+Na)⁺. Amino acid analysis: Val, 0.95: Leu, 1.02; Phe, 1.02;Tyr(O-AcOH), present.

EXAMPLE 4 H-Tyr(O-AcOH)-Val-ACHPA-Leu-Phe-NH₂.HCl

A solution of Boc-Tyr(O-AcOH)-Val-ACHPA-Leu-Phe-NH₂ (25 mg, 0.028 mmol)in 6N HCl/dioxane (1.5 mL) was stirred at room temperature under anitrogen atmosphere for 20 min. The solvent was evaporated and theresidue was dried under reduced pressure for 1 h. The solid wastriturated with Et₂ O and the resulting suspension was filtered. Thecollected precipitate was dried under reduced pressure to afford thetitle compound (20 mg, 86%). Mass spectrum: 795 (M-Cl)⁺. Amino acidanalysis: Val, 0.94; Leu, 1.02; Phe, 1.04; Tyr(O-AcOH), present.

EXAMPLE 5 Boc-Tyr(O-AcOH)-Phe-ACHPA-Leu-Phe-NH₂

The title compound was prepared by the procedures described in examples1 to 3 but substituting Boc-Phe-OH for Boc-Val-OH. Mass spectrum: 943(M+H)⁺. Amino acid analysis: Leu, 0.99; Phe, 2.01; ACHPA, 0.86;Tyr(O-AcOH), 0.86.

Other examples of peptides of formula 1 include: ##STR4##H-Tyr-(O-AcOBzl)-Ala-ACHPA-Leu-Phe-NHC(CH₂ OH)₂ CH₃H-Tyr-(O-AcOH)-(N-Me)Asp-ACHPA-Cha-Asn-NH₂

H-(3-iodo)-Tyr(O-AcOBzl)-Val-ACHPA-Leu-Phe-NH₂

H-(3,5-diiodo)-Tyr(O-AcOBzl)-Val-ACHPA-Leu-Phe-NH₂

Boc-Tyr(O-AcOEt)-Ala-AHPPA-Gln-Ile-OH

H-Tyr(O-AcOH)-Cpa-ACHPA-Tyr-NHCH₃

Boc-Tyr(O-AcOH)-Val-ACHPA-NHC(CH₂ OH)₂ CH₃

EXAMPLE 6

Recombinant HIV Protease HPLC Assay:

Enzyme: HIV protease was expressed in E. coli and purified to ca. 50%purity according to the procedure described by H.-G. Krausslich et al.,Proc. Natl. Acad. Sci. USA, 86, 807 (1989). The enzyme was stored as 10μL aliquots at -70° C. The aliquots were diluted to 1/10th of theoriginal concentration with buffer prior to use (enzyme workingsolution).

Substrate: VSFNFPQITL-NH₂, MW 1164, see Krausslich et al., supra, wasused as substrate. The substrate was made into 10 mM stock in DMSO andstored at -20° C. Prior to use, the stock was diluted with buffer togive a 400 μM solution substrate working solution.

Buffer: 2-(4-Morpholino)ethanesulfonic acid (50 mM), NaCl (25 mM) andEDTA (5 mM) was dissolved in distilled H₂ O (90 mL) and the solution wasadjusted to pH6 with concentrated aqueous NaOH. The latter solution wasdiluted to 100 mL with H₂ O to give the buffer.

Procedure: (1) The test compound was dissolved in DMSO to give asolution having 40X the final concentration of the test compound in theassay mixture (see step 2) so that the amount of DMSO in the assaymixture was 5% (v,v) or less. (2) The assay mixture was prepared bymixing 10 μL of the substrate working solution, 0.5 μL of the solutionof the test compound in DMSO from step 1, and 10 μL of the enzymeworking solution. (3) The assay mixture was incubated at 37° C. for 1 h.(4) The reaction was quenched by adding 100 μL of 2% aqueous TFA. (5)The substrate and products (i.e. VSFNF and PQITL-NH₂) were separated bysubjecting 100 μL of the quenched assay mixture to HPLC using Nucleosil®C₁₈ column with a 26-min linear gradient at 1 mL/min from 11% to 70%acetonitrile in H₂ O with 0.05% aqueous TFA. Elution was monitored at210 nm. (6) A control which was the assay mixture without the testcompound, was subjected simultaneously to steps 3 to 5.

Inhibition Studies: Cleavage products and remaining parent substratewere quantified by either peak height or by integration of theappropriate HPLC peaks. Substrate conversion was calculated using thefollowing relationship: ##EQU1## Enzyme inhibition of the test compoundwas calculated as follows: ##EQU2##

The concentration of the test compound which causes a 50% inhibition ofthe HIV-protease, i.e. the IC₅₀, was determined as follows:

The percent inhibition of the enzyme was determined for a minimum ofthree different concentrations of the test compound. Thereafter, theIC₅₀ was determined graphically by plotting the percent inhibition ofthe substrate against the concentration of the test compound.

The following table of exemplified peptides of formula 1 lists theirIC₅₀ as determined in the recombinant HIV protease HLPC assay.

    ______________________________________                                                               Example in                                                                    which pep-                                                                    tide is pre-                                                                            IC.sub.50                                    Peptide                pared     (nM)                                         ______________________________________                                        Boc-Tyr(O--AcOBzl)-Val-ACHPA-Leu-                                                                    1         70                                           Phe-NH.sub.2                                                                  H-Tyr(O--AcOBzl)-Val-ACHPA-Leu-Phe-                                                                  2          5                                           NH.sub.2.HCl                                                                  Boc-Tyr(O--AcOH)-Val-ACHPA-Leu-                                                                      3         52                                           Phe-NH.sub.2                                                                  H-Tyr(O--AcOH)-Val-ACHPA-Leu-Phe-                                                                    4         10                                           NH.sub.2.HCl                                                                  Boc-Tyr(O--AcOH)-Phe-ACHPA-Leu-                                                                      5         1000                                         Phe-NH.sub.2                                                                  ______________________________________                                    

What is claimed is:
 1. A peptide selected from the group consistingof:Boc-Tyr(O-AcOBzl)-Val-ACHPA-Leu-Phe-NH₂H-Tyr(O-AcOBzl)-Val-ACHPA-Leu-Phe-NH₂Boc-Tyr(O-AcOH)-Val-ACHPA-Leu-Phe-NH₂H-Tyr(O-AcOH)-Val-ACHPA-Leu-Phe-NH₂.Boc-Tyr(O-AcOH)-Phe-ACHPA-Leu-Phe-NH₂
 2. A pharmaceutical compositioncomprising a peptide as recited in claim 1, or a therapeuticallyacceptable salt thereof, and a pharmaceutically acceptable carrier.